A method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential enrichment, termed the SELEX process, is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled "Systematic Evolution of Ligands by EXponential Enrichment," now abandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled "Nucleic Acid Ligands," now U.S. Pat. No. 5,475,096, U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled "Methods for Identifying Nucleic Acid Ligands," now U.S. Pat. No. 5,270,163 (see also WO 91/19813), each of which is herein specifically incorporated by reference. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule. The SELEX process provides a class of products which are referred to as nucleic acid ligands, such ligands having a unique sequence, and which have the property of binding specifically to a desired target compound or molecule. Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule. SELEX is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size can serve as targets.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
It has been recognized by the present inventors that the SELEX method demonstrates that nucleic acids as chemical compounds can form a wide array of shapes, sizes and configurations, and are capable of a far broader repertoire of binding and other functions than those displayed by nucleic acids in biological systems.
The dogma for many years was that nucleic acids had primarily an informational role. Through the application of SELEX, it has become clear to the present inventors that nucleic acids have three dimensional structural diversity not unlike proteins. As such, the present inventors have recognized that SELEX or SELEX-like processes could be used to identify nucleic acids which can facilitate any chosen reaction in a manner similar to that in which nucleic acid ligands can be identified for any given target. In theory, within a candidate mixture of approximately 10.sup.13 to 10.sup.18 nucleic acids, the present inventors postulate that at least one nucleic acid exists with the appropriate shape to facilitate a broad variety of physical and chemical interactions.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled "Method for Selecting Nucleic Acids on the Basis of Structure," now abandoned, describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled "Photoselection of Nucleic Acid Ligands," now abandoned, describes a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled "High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine," abandoned in favor of U.S. patent application Ser. No. 08/443,957, now U.S. Pat. No. 5,580,737, describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed Counter-SELEX. U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled "Systematic Evolution of Ligands by EXPONENTIAL Enrichment: Solution SELEX," abandoned in favor of U.S. patent application Ser. No. 08/461,069, now U.S. Pat. No. 5,567,588, describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides," abandoned in favor of U.S. patent application Ser. No. 08/430,709, now U.S. Pat. No. 5,660,985, that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines. U.S. patent application Ser. No. 08/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'-NH.sub.2), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled "Novel Method of Preparation of Known and Novel 2'-Modified Nucleosides by Intramolecular Nucleophilic Displacement," describes oligonucleotides containing various 2'-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. patent application Ser. No. 08/284,063, filed Aug. 2, 1994, entitled "Systematic Evolution of Ligands by EXponential Enrichment: Chimeric SELEX", now U.S. Pat. No. 5,637,459, and U.S. patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled "Systematic Evolution of Ligands by EXponential Enrichment: Blended SELEX," now U.S. Pat. No. 5,683,867 respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules. Each of the above described patent applications which describe modifications of the basic SELEX procedure are specifically incorporated by reference herein in their entirety.
Previous efforts have demonstrated the use of SELEX to identify ligands that can act facilitatively. In co-pending application, International Publication No. WO 95/08003, filed Sep. 16, 1994 which is a Continuation in Part of U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled "Photoselection of Nucleic Acid Ligands," specifically incorporated by reference, certain nucleic acid sequences that contained 5-iodouracil residues were identified that covalently bind to HIV-1 Rev protein. In co-pending patent application U.S. application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX," now U.S. Pat. No. 5,683,867, specifically incorporated by reference, a nucleic acid ligand to human neutrophil elastase was identified wherein a functional unit was associated with the nucleic acid ligand. In this instance, the functional unit was a valyl phosphonate that bound covalently to the elastase target. In co-pending patent application U.S. application Ser. No. 08/309,245, filed Sep. 20, 1994, entitled "Parallel SELEX," now U.S. Pat. No. 5,723,289, specifically incorporated herein by reference, the covalent reaction between a pool of reactants to form a product library is specifically facilitated by a nucleic acid attached to certain reactants.
Ribonucleic acid (RNA) is a biopolymer that is integral to protein synthesis and cellular metabolism. RNA is primarily composed of four basic monomers interlinked with phosphate diester linkages to form a directional (5'-3') polymer. In nature RNA molecules are usually transcribed as single stranded and may fold in various lengths and shapes, ranging from short oligoribonucleotide primers for DNA synthesis to the very large ribosomal RNAs which form part of the translational machinery active during protein synthesis. The purine and pyrimidine bases of RNA interact via ring-stacking and hydrogen bonding networks to fold the polymer chains into compact structures containing surfaces capable of providing specific binding sites for a variety of ligands, including proteins, nucleic acids, divalent metal ions, and carbohydrates (Saenger (1984) in Principles of Nucleic Acid Structure (C. R. Cantor, ed.) Chapters 6, 8, and 10, Spinger-Verlag, New York, N.Y.). Single stranded RNA (ssRNA) molecules commonly form localized regions of secondary structure, such as hairpin loops and pseudoknot structures, by base-pairing between complementary regions of the same molecule. (Schimmel (1989) Cell 58:9). The 2'-hydroxyl group provides additional hydrogen bonding capacity and serves as a modest nucleophile in the presence of electropositive magnesium ions. In fact, some RNAs are capable of topologically juxtaposing surface recognition and catalysis elements to promote metal co-factor driven chemical reactions such as transesterification and phosphodiester bond hydrolysis (Cech (1987) Science 236:1532-1539). These sophisticated attributes have prompted investigators to pursue therapeutic applications based on RNA catalysis and specific ligand binding activities.
Studies to date have identified only a few nucleic acids which have only a narrow subset of facilitating capabilities. A few RNA catalysts are known (Cech (1987) Science 236:1532-1539; McCorkle and Altman (1987) Journal of Chemical Education 64:221-226). These naturally occurring RNA enzymes (ribozymes) have to date been shown to act primarily on oligonucleotide substrates. Further, these molecules perform over a narrow range of chemical possibilities, which are thus far related primarily to phosphodiester bond condensation/hydrolysis, with the exception of the possible involvement of RNA in protein biosynthesis. Despite intense recent investigation to identify RNA or DNA catalysts, few successes have been identified. Phosphodiester cleavage (Beaudry and Joyce (1992) Science 257:635), hydrolysis of aminoacyl esters (Piccirilli et al. (1992) Science 256:1420-1424); self-cleavage (Pan and Uhlenbeck (1992) Biochemistry 31:3887), RNA polymerase activity (an RNA polymerase is an enzyme which facilitates the ligation of the 3' OH of one RNA molecule with the 5' triphosphate end of a second RNA molecule) (Bartel and Szostak (1993) Science 261:1411-1418); biphenyl isomerase activity (Prudent et al. (1994) Science 264:1924-1927); and polynucleotide kinase activity (a kinase is an enzyme which facilitates the transfer of a phosphate from one substrate to another) (Lorsch and Szostak (1994) Nature 371:31-36) have been observed. Dai et al. (1995) Science 267:237-240, have identified an RNA ligand which facilitates the cleavage of an alkyl amide.
The nucleic acid catalysts identified to date have certain shortcomings associated with their effectiveness in bond forming/breaking reactions. Among the drawbacks are that they act slowly relative to protein enzymes, and as described above, they perform over a somewhat narrow range of chemical possibilities.
Transfer RNA (tRNA) is a type of RNA molecule which acts as an adaptor during protein synthesis, matching amino acids (aa) to their codons on messenger RNA (mRNA). Before an amino acid is incorporated into a protein chain, it is attached by its carboxyl end to the 3' end of a tRNA molecule. This reaction is facilitated in two steps by a class of enzymes referred to as aminoacyl-tRNA synthetases, as illustrated in equations (1) and (2). In the first step, equation (1), aminoacyl-tRNA synthetase activates the carbonyl group of the amino acid (aa) by forming the aminoacyl-adenylate, in which the carboxyl group of the amino acid is attached to adenosine monophosphate (AMP): ##STR1## The enzyme-bound aa-AMP intermediate is then transferred to the 2' or 3' hydroxy terminus of a tRNA molecule, thereby forming an aa-tRNA molecule and adenosine monophosphate (AMP) (Equation (2)): ##STR2## A tRNA molecule carrying its cognate amino acid is said to be "charged". The enzyme bound adenylate therefore, facilitates the esterification of the 2'(3') hydroxy terminus of transfer RNA, to create an aminoacyl-tRNA molecule.